Liver tumor marker sequences

ABSTRACT

Polypeptides whose expression is upregulated in liver tumor cells and cells from liver preneoplastic foci relative to expression in normal liver cells are disclosed as are polynucleotides that encode the polypeptides. In humans, the polynucleotide maps to a region of chromosome 15. The overexpression has also been confirmed in human liver, breast, colon and kidney cancer cell lines. It is believed that the polypeptides are overexpressed in tumor and preneoplastic cells in general.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. provisional applicationSerial No. 60/396,626, filed on Jul. 17, 2002, which is hereinincorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with U.S. Government Support from thefollowing agency: NIH, Grant No. CA22484. The U.S. Government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] Primary liver cancer is the fifth most common cancer worldwidewith approximately half a million cases reported in 1990. Hepatocellularcarcinoma (HCC) accounts for 80% of all liver cancer and the rates ofHCC have increased by over 70% in the last two decades in the U.S. Thefatality ratio (mortality/incidence) of liver cancer is approximately 1,indicating that the majority of patients live less than a year. Latediagnosis due to lack of clinical symptoms is one of the main reasonsfor the high fatality ratio.

[0004] Liver cancer can result from both viral infection and chemicalexposure. Known risk factors include hepatitis B and C virus infectionand exposure to aflatoxin β1. It is not known whether distinct routes toliver cancer affect the same or different cellular pathways. Nomutational model has yet been developed for liver cancer as it has beenfor other cancers such as colon cancer. The molecular events thatprecede neoplastic transformation of the liver are not well understood.With no clearly identified cause, successful treatment options arelacking. In fact, the specific genes that are deregulated in livercancer have not yet been enumerated. This is a critical first step indeveloping a successful strategy for treating liver cancer.

[0005] There is a pressing need to understand the molecular eventsassociated with the development of liver cancer, both in humans and inanimal model systems where liver cancer is extensively studied, and toprovide diagnostic and therapeutic reagents for treating same.

BRIEF SUMMARY OF THE INVENTION

[0006] The invention is summarized in that polypeptides of the inventionare found in liver tumor cells and in cells from preneoplastic liverfoci in human and non-human animals at levels higher than are found inregenerating or quiescent normal liver tissue. This finding has beenconfirmed in human breast, colon and kidney cancer cell lines. As aresult of this differential overexpression, the polypeptides, as wellsas polynucleotides that encode the polypeptides, are diagnostic markersfor cancer in general, especially liver, breast, colon and kidneycancer, in a human or non-human animal.

[0007] In one aspect, the present invention relates to an isolatedpolypeptide containing an amino acid sequence of SEQ ID NO:2, an aminoacid sequence that is at least 70% identical to SEQ ID NO:2 over thelength of SEQ ID NO:2, an amino acid sequence of amino acid 22 to aminoacid 439 of SEQ ID NO:2 (secreted portion of SEQ ID NO:2), an amino acidsequence that is at least about 68% identical to amino acid 22 to aminoacid 439 of SEQ ID NO:2 over the length of amino acid 22 to amino acid439 of SEQ ID NO:2, an amino acid sequence of SEQ ID NO:4, an amino acidsequence that is at least 70% identical to SEQ ID NO:4 over the lengthof SEQ ID NO:4, an amino acid sequence of amino acid 22 to amino acid400 of SEQ ID NO:4 (secreted portion of SEQ ID NO:4), an amino acidsequence that is at least about 68% identical to amino acid 22 to aminoacid 400 of SEQ ID NO:4 over the length of amino acid 22 to amino acid400 of SEQ ID NO:4. The percentage identity of sequences is determinedusing the Blosum62 alignment method.

[0008] In another aspect, the invention also relates to an isolatednucleic acid containing a polynucleotide that encodes a polypeptide ofthe invention, to a complement of the polynucleotide, or to apolynucleotide that is at least about 80% identical, more preferably 90%identical, and still more preferably 95% identical to an aforementionedpolynucleotide of the invention, using the Wilbur-Lipman DNA Alignmentmethod. A polynucleotide that encodes a polypeptide of the invention caninclude but is not limited to SEQ ID NO:1 from nucleotide 25 tonucleotide 1341, which encodes SEQ ID NO:2, as well as SEQ ID NO:3 fromnucleotide 1 to nucleotide 1200, which encodes SEQ ID NO:4. SEQ ID NO:3,predicted by the inventors to represent a coding region on humanchromosome 15 (contig Hs15_(—)10351), is 82.4% identical to thepolypeptide-encoding portion of SEQ ID NO:1 using the Wilbur-Lipman DNAAlignment method.

[0009] In another aspect, a polynucleotide of the invention isengineered into a genetic construct downstream from a heterologouspromoter not natively upstream of the polynucleotide that directstranscription of the polynucleotide. The genetic construct is introducedinto a host cell that supports transcription of the polynucleotide andtranslation of the encoded polypeptide which can then be purified usingmethods known to those skilled in the art. Alternatively, the constructcomprising a polynucleotide of the invention is provided in an in vitrotranscription/translation system for producing the encoded polypeptide.

[0010] In yet another aspect, the present invention provides a host celltransfected with a genetic construct of the invention.

[0011] In still another aspect, the invention is an antibody thatspecifically binds to a polypeptide of the invention.

[0012] In yet another aspect, the invention is a method for identifyingan agent that modulates the expression of a polypeptide of the invention(e.g., an inducer or suppressor). The method includes the steps ofexposing a cell that contains a polynucleotide of the invention underthe control of its native promoter, measuring the expression of thepolynucleotide in the cell, and comparing the expression to that in acontrol cell that is not exposed to the test agent. A higher or lowerthan the expression in the control cell indicates that the agent canmodulate the expression of the polynucleotide. The expression can bemeasured and compared at either the mRNA level or the protein level.Preferably, a liver, breast, colon or kidney cell (cancerous or normal)is used in the method. More preferably, a human or murine liver, breast,colon or kidney cell is used.

[0013] In still another aspect, the present invention is a method ofdiagnosing cancer or preneoplastic development in a tissue or organ of ahuman or non-human animal by measuring the expression of a polypeptideof the invention in cells of the tissue or organ obtained from a regionsuspected of cancer or preneoplastic development, and comparing theexpression to a normal standard, wherein a higher than normal expressionindicates cancer or preneoplastic development in the suspected region. Askilled artisan can readily establish a normal standard. For example, itcan be the expression level in normal cells of the same tissue or organin the same animal, or it can be an expression level range establishedby testing normal cells of the same tissue or organ of other animals ofthe same species. The expression can be measured and compared at eitherthe mRNA level or protein level.

[0014] In a related aspect, the present invention is a method foridentifying a candidate human or non-human animal for further cancerscreening, where the method includes, in one embodiment, the step ofdetermining the level of a polypeptide of the invention in a blood orblood-derived sample from the animal, whereby the animal is identifiedas a candidate for further cancer screening when the level exceedseither a normal range established by the same animal during a periodthat is tumor-free in the tissue or organ, or a normal range establishedby other animals of the same species that are tumor-free in the tissueor organ. In another embodiment, the method takes advantage of theexpected secretion of the polypeptide and the development of antibodiesto the polypeptide in a human or non-human animal that overexpresses thepolypeptide in the cancerous or preneoplastic tissue or organ. Themethod includes the step of determining the level of an antibody to thepolypeptide in a blood or blood-derived sample from the animal, wherebythe animal is identified as a candidate for further cancer screeningwhen the antibody level exceeds either a normal range established by thesame animal during a period that is tumor-free in the tissue or organ,or a normal range established by other animals of the same species thatare tumor-free in the tissue or organ. It is understood that individualsfree of cancer or preneoplastic development in the tissue or organ maynot develop an antibody to the polypeptide. Thus, the normal range forthe level of the antibody can be zero.

[0015] In still another aspect, the invention relates to a kit suitablefor use in a method for determining the level of a polypeptide orpolynucleotide of the invention, where the kit contains at least one ofan antibody specifically directed to an epitope on a polypeptide of theinvention and a polynucleotide that hybridizes to a polynucleotide ofthe invention, as well as at least one control sample component forwhich the relative or absolute amount of the polynucleotide orpolypeptide of the present invention is known, the control samplecomponent being selected from liver cancer cells, preneoplastic livercells, normal liver cells, breast cancer cells, normal breast cells,colon cancer cells, normal colon cells, kidney cancer cells, normalkidney cells, an extract of any of the foregoing cells, a blood samplefrom a human or non-human animal, and a blood-derived sample from ahuman or non-human animal.

[0016] It is an object of the present invention to provide apolynucleotide and a polypeptide that are differentially expressed inpreneoplastic or cancer cells and normal regenerating or quiescent cellsin a tissue or organ of a human and non-human animal.

[0017] Other objects, features and advantages of the present inventionwill become apparent upon consideration of the following detaileddescription of the invention.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0018]FIG. 1 shows the cloning of CRG-L2. a) RT-PCR analysis of CRG-L2in mouse liver tissues. Because the C3H/HeJ mice used in these studiesare inbred, all untreated mice are genetically identical. Accordingly,we have never observed any differences in CRG-L2 expression incomparison of individual normal mice. Therefore, quiescent,regenerating, and newborn RNA samples were prepared from several miceand then pooled so that the same pooled RNA samples could be used inmultiple experiments. Quiescent and regenerating samples are acombination of four livers and newborn samples are a combination ofeight livers. However, it is known that tumors display heterogeneousgenetic and molecular profiles. Therefore, to take into considerationthese possible differences, the tumor samples used in our experimentsare from individual mice. b) mRNA structure of CRG-L2. Alignment of the5′ and 3′ RACE products suggest that CRG-L2 mRNA can contain one ofthree alternative 3′UTRs. c) Northern blot hybridization of CRG-L2 inquiescent liver and four individual liver tumors. Four bands weredetected at 2.4, 3.0, 5.5, and 10 kb mRNAs. The three smaller mRNAscorrespond to clones A, B, and C. A fourth band, D, was not clonedprobably due to inefficient PCR through a long 3′ UTR. d) The CRG-L2open reading frame was aligned to mouse chromosome 9 (31 cM). Exons arerepresented by black boxes. The distance between some of the exons isestimated since there are gaps between the contigs in the genome andthese gaps are represented by a >sign. CRG-L2 is localized withinchromosome 15q21.2 of the human genome and a similar intron/exonstructure is suggested by comparing the mouse cDNA to the human genome.

[0019]FIG. 2 shows structural features of the CRG-L2 protein. a) CRG-L2cDNA encodes a protein of 48 kD containing two collagen domains and anolfactomedin domain. b) Hydrophobicity analysis of the putative CRG-L2protein with the Kyle-Doolittle algorithm. Positive values representhydrophilic regions and negative values represent hydrophobic regions.

[0020]FIG. 3 shows that CRG-L2 expression is increased in humanhepatocellular carcinomas. Top panel is a phosphoimage of the RT-PCRresults measuring CRG-L2 mRNA and middle panel is a longerautoradiographic exposure. Equal loading was confirmed by analysis ofGAPDH mRNA. All HCC were classified as moderately differentiated.

[0021]FIG. 4 shows that CRG-L2 expression is restricted in normaltissues. CRG-L2 mRNA was amplified in multiple mouse (a) and human (b)tissues using Multiple Tissue cDNA Panels. Aliquots of the PCR productswere taken out at the indicated cycles.

DETAILED DESCRIPTION OF THE INVENTION

[0022] Liver cancer is generally studied in animal model systems,preferably in rodent systems, where certain strains are bred for highsusceptibility to liver tumors. C3H/HeJ mice are highly susceptible toliver tumors after induction with diethylnitrosamine (DEN). To identifypolynucleotide sequences or genes that show differential expression inliver tumor cells as compared to normal liver cells, gene expressiondifferences between liver tumors and a regenerating liver weredetermined using representational difference analysis (RDA: Lisitsyn, etal., Science 259:946 (1993), incorporated by reference as if set forthherein in its entirety).

[0023] In this application, the applicants disclose polypeptides frommurine animals (SEQ ID NO:2) and humans (SEQ ID NO:4) that areupregulated in cells and cell extracts from human and murine livertumors and liver preneoplastic tissues, relative to quiescent andregenerating normal liver cells. The polypeptide is therefore given thename human or murine Cancer Related Gene-Liver 2 (CRG-L2). CRG-L2overexpression was also found in human liver, breast, colon and kidneycancer cell lines. Thus, despite of its name indicative of liver origin,it is believed to be overexpressed in other types of cancer andpreneoplastic cells in general, especially breast, colon and kidneycancer and preneoplastic cells.

[0024] Using the Blosum62 alignment method, the human and murine CRG-L2sare found to be 76% identical. It is expected that CRG-L2s from otheranimals, e.g., other mammals, are at least 70% identical to either thehuman or murine CRG-L2 if compared using the same alignment method.Hydrophobic sequences are present within the first 30 amino acids of SEQID NO:2 and SEQ ID NO:4. Based on information obtained from otherproteins with leader sequences, the serine at amino acid position 21 ofboth SEQ ID NO:2 and SEQ ID NO:4 is believed to be the cleavage site ofa leader sequence for the secretion of both of the CRG-L2s. Accordingly,it is believed that when amino acids 1-21 are cleaved, the remainingamino acid sequences of SEQ ID NO:2 and SEQ ID NO:4 can be secreted fromcells. Corresponding leader sequences on other CRG-L2s can be readilyidentified by a skilled artisan. Depending on the variability of theleader sequences among CRG-L2s, the percentage of identity among thesecreted sequences may be about 3% higher or lower than the overall 70%identity. Generally speaking, it is expected that the secreted portionof CRG-L2s in other animals, e.g., other mammalians, are at least about68% identical to either the secreted portion of the human or that of themurine CRG-L2.

[0025] Also disclosed are polynucleotides that encode the polypeptidesof the invention (e.g., the full length and the secreted CRG-L2s), whichcan include, without limitation, mRNA, single or double stranded DNA,cDNA and the like. In addition to the primary murine cDNA productdisclosed as SEQ ID NO:1, two additional variant murine cDNAs that arebelieved to derive from alternative 3′ untranslated regions were alsoobtained. The variant murine cDNA molecules differ from SEQ ID NO:1 inthe 3′ untranslated portion of the molecules, commencing respectively atnucleotide 1937 and at nucleotide 2342, as shown in the SequenceListing. SEQ ID NO:3 discloses a sequence from human Chromosome 15 thatencodes the human CRG-L2 of SEQ ID NO:4.

[0026] Further, the invention provides materials and methods fordetecting expression (and changes in expression) of the polypeptides andof the polynucleotides that encode the polypeptides, therebyfacilitating use as a diagnostic marker for cancer and preneoplasticdevelopment and as a system for assessing putative therapeutic agents.As described in detail in the example below, since the CRG-L2 eitherbelongs or is similar to the family of cancer-testis antigens, it isexpected that a patient will display an immune response to CRG-L2 whenit is overexpressed in preneoplastic and cancerous tissues. Therefore,detecting or measuring the level of an antibody to CRG-L2 in a blood orblood-derived sample from a patient provides another diagnostic tool.

[0027] Structurally, the murine CRG-L2 protein (SEQ ID NO:2) contains439 amino acids and has a predicted molecular weight of about 47.5 kDA.Using the Simple Modular Architecture Research Tool(http://smart.embl-heidelberg.de/), it was determined that the murineCRG-L2 includes two collagen domains in the 5′ region (corresponding toamino acids 29-88 and 89-149 of SEQ ID NO:2, respectively) and a largeolfactomedin domain near the C-terminus (corresponding to amino acids189-433 of SEQ ID NO:2). The human protein also contains two putativecollagen domains and one olfactomedin domain at amino acids 27-85,86-145, and 177-395 of SEQ ID NO:4, respectively. Olfactomedin-relatedproteins are secreted glycoproteins having conserved C terminal motifs.It is anticipated that CRG-L2 can be secreted into the blood and anincrease in blood CRG-L2 level over normal levels is diagnostic ofcancer and preneoplastic development. Preferably, the diagnostic bloodCRG-L2 level is set to be at least about 5%, more preferably at leastabout 10%, and most preferably at least about 25% over a normal level.

[0028] The term “isolated nucleic acid” or “isolated polypeptide” usedin the specification and claims of the present invention means a nucleicacid or polypeptide isolated from its natural environment or preparedusing synthetic methods such as those known to one of ordinary skill inthe art. Complete purification is not required in either case. Aminoacid and nucleotide sequences that flank a polypeptide or polynucleotidethat occurs in nature, respectively, can but need not be absent from theisolated form. The polypeptides and nucleic acids of the invention canbe isolated and purified from normally associated material inconventional ways such that in the purified preparation the polypeptideor nucleic acid is the predominant species in the preparation. At thevery least, the degree of purification is such that the extraneousmaterial in the preparation does not interfere with use of thepolypeptide or nucleic acid of the invention in the manner disclosedherein. The polypeptide or nucleic acid is preferably at least about 85%pure, more preferably at least about 95% pure and most preferably atleast about 99% pure.

[0029] Further, an isolated nucleic acid has a structure that is notidentical to that of any naturally occurring polynucleotide or to thatof any fragment of a naturally occurring genomic polynucleotide spanningmore than three separate genes. An isolated nucleic acid also includes,without limitation, (a) a polynucleotide having a sequence of anaturally occurring genomic or extrachromosomal nucleic acid moleculebut which is not flanked by the coding sequences that flank the sequencein its natural position; (b) a polynucleotide incorporated into a vectoror into a prokaryote or eukaryote genome such that the resultingmolecule is not identical to any naturally occurring vector or genomicDNA; (c) a separate molecule such as a cDNA, a genomic fragment, afragment produced by polymerase chain reaction (PCR), or a restrictionfragment; and (d) a recombinant nucleotide sequence that is part of ahybrid gene, i.e., a gene encoding a fusion protein. Specificallyexcluded from this definition are polynucleotides present in mixtures ofclones, e.g., as these occur in a DNA library such as a cDNA or genomicDNA library. An isolated nucleic acid can be modified or unmodified DNAor RNA, whether fully or partially single-stranded or double-stranded oreven triple-stranded. A nucleic acid can be chemically or enzymaticallymodified and can include so-called non-standard bases such as inosine.

[0030] The nucleotide sequences of the invention can be introduced into,and expressed in, host cells which can be prokaryotic (such asbacterial) cells or eukaryotic (such as yeast, insect, amphibian ormammalian) cells whereupon the transcription of polynucleotide and theproperties of the encoded polypeptides can be assessed.

[0031] The disclosure of the CRG-L2 sequences that are upregulated inliver tumor and preneoplastic cells, and in human breast, colon andkidney cancer cell lines provides a means for identifying (in vivo or invitro) candidates for further testing as preventive and therapeuticagents. For example, animal cells that contain a CRG-L2 sequence underthe control of its native promoter can be exposed to a test agent andthe effect of the test agent on the CRG-L2's expression at the mRNA orprotein level relative to that of untreated controls can be measured.Alternatively, the level of expression can be assessed in biologicalsamples taken directly from a human or non-human tissue. Presumably, ananti-tumor agent can bring down the mRNA and protein level in tumorcells. Accordingly, an agent that demonstrates such an activity is agood candidate for further testing for anti-tumor efficacy.

[0032] The presence and level of such a differentially expressed proteincan be readily discerned using antibodies directed to an epitope on theprotein using well known methods, such as an ELISA method. It is wellwithin the skill of one of ordinary skill in the art to generate suchantibodies. The presence and level of mRNA for the protein can bemeasured using methods for hybridizing nucleic acids (including, withoutlimitation, RNA, DNA, and cDNA). Such methods are generally known tothose skilled in the art, but are enabled by the disclosure herein of atumor-specific sequence. Examples of such methods include but are notlimited to RT-PCR amplification, Northern blot and Southern blot.

[0033] Given the disclosure herein of polynucleotides that encode CRG-L2of human, murine and other animal species, one of ordinary skill in theart knows how to design primers for use in RT-PCR analysis and probesfor Northern and Southern blot. The Example below describes a method ofusing RT-PCR to measure CRG-L2 mRNA level in liver tumor cells, liverpreneoplastic cells and normal liver cells. The RT-PCR amplified afragment of CRG-L2 cDNA (SEQ ID NO: b 1) and its noted 3′ end variants,and the mRNA level in liver tumor and preneoplastic cells was observedto be higher than that in normal liver cells. Accordingly, a suitableCRG-L2 sequence for amplifying or probing in analyzing differentialCRG-L2 mRNA levels is one that corresponds to a fragment shared by allthree CRG-L2 cDNA sequences. A CRG-L2 mRNA sequence that corresponds toa fragment unique to the longer 3′ untranslated sequence variants couldalso be used to analyze differential CRG-L2 mRNA expression sinceNorthern analysis has shown that all three mRNAs are differentiallyexpressed in liver tumor and preneoplastic cells relative to normalliver cells.

[0034] A skilled artisan understands that the polynucleotides disclosedherein can contain additional nucleotides at the 5′-end, 3′-end or boththat do not affect the function of the polynucleotides in terms of theiruses contemplated herein. The additional nucleotides can but do not haveto assist in the cloning, detection and purification proceduresassociated with the use of the polynucleotides. Similarly, a skilledartisan understands that the polypeptides disclosed herein can containadditional amino acid sequences at the N- or C-terminus or both that donot affect the function of the polypeptides. The additional amino acidsequences can but do not have to assist in purification, detection, orstabilization of the polypeptides.

[0035] Further, a skilled artisan understands that polynucleotide andpolypeptide sequences presented herein can vary somewhat, whether as aresult, e.g., of sequencing error or allelic variation or duplication,from the sequence presented while still retaining their essentialnature, that is, higher expression level in tumor and preneoplasticcells relative to normal cells. Further, the polynucleotides of theinvention include conservatively modified variants of the sequencespresented herein, complementary sequences, and splice variants. In viewof the known degeneracy in the genetic code, the proteins orpolypeptides disclosed can also be encoded by a large number of otherpolynucleotide sequences, all of which are within the scope of theinvention. Polynucleotide sequences that are at least 80% identical tothe polynucleotide sequences that encode the polypeptide sequencesdisclosed herein can be used as hybridization probes for codingsequences and are thus within the scope of the present invention. Thepolynucleotides and polypeptides of the invention include, withoutlimitation, polymorphic variants, alleles, mutants, and interspecieshomologs that (1) are expressed at higher level in tumor andpreneoplastic cells, especially in liver, breast, colon and kidney tumorand preneoplastic cells, (2) bind to antibodies raised against thecoding region of the disclosed polypeptides, (3) specifically hybridizeunder stringent or moderately stringent hybridization conditions to apolynucleotide that encodes a polypeptide of the present invention, or(4) are amplified by primers that amplify a polynucleotide that encodesa polypeptide of the present invention.

[0036] Exemplary stringent hybridization conditions include 50%formamide, 5×SSC and 1% SDS incubated at 42° C., or 5×SSC and 1% SDSincubated at 65° C., followed by washing in 0.2×SSC and 0.1% SDS at 65°C. Exemplary moderately stringent hybridization conditions include 40%formamide, 1M NaCl and 1% SDS incubated at 37° C. followed by washing in1×SSC at 45° C. These conditions are merely exemplary as one skilled inthe art is readily able to discern stringent from moderately stringenthybridization conditions.

[0037] Moreover, the sequences of the invention also encompasssubstitutions, additions and deletions of the sequences presented wherethe change affects one or a few amino acids in the presented polypeptidesequences, without substantial effect upon the activity of thepolypeptide, i.e., differential expression in cancer cells andpreneoplastic cells relative to normal cells.

[0038] The present invention will be better understood uponconsideration of the following non-limiting example.

EXAMPLE Materials and Methods

[0039] Rapid Amplification of cDNA Ends (RACE). Rapid amplification ofcDNA ends (RACE) was performed in both directions using the SMART cDNAamplification kit (Clontech) from mouse liver tumor polyA RNA. 5′ and 3′RACE were performed using the gene-specific primers, GSP-A[5′-GCATGGCAAGAACAGACTGG-3′] (SEQ ID NO:5) and GSP-B[5′-GGATGAGAAGGGCATCTGGA-3′] (SEQ ID NO:6). 5′ and 3′ RACE products thatwere identified with the corresponding GSP primer were gel extracted andcloned into TOPO-TA vector (Invitrogen). Cloned products were sequencedby Big Dye (ABI) in the McArdle Laboratory Sequencing Facility(University of Wisconsin-Madison).

[0040] RNA Analysis. For analysis of murine CRG-L2 mRNA, total RNA wasextracted from liver using guanidine thiocyanate/CsCl as describedpreviously in (Lukas et al., 1999, incorporated by reference in itsentirety). PolyA mRNA was isolated from 250 μg of total RNA usingOligotex mRNA Kit (Qiagen). RT-PCR was performed as described previously(Graveel et al., 2001, incorporated by reference in its entirety) withprimers, RDA-3a [5′-CAACAACCTGGCTTAGAGC-3′] (SEQ ID NO:7) and RDA-3b[5′-GCCATCTGATGCTCTATCC-3′] (SEQ ID NO:8).

[0041] For Northern blot hybridization, polyA RNA samples (2 μg) wereprepared and electrophoresed as described previously (Lukas et al.,1999). Gel was soaked in 5 volumes of water for 5 min and thentransferred overnight to a GeneScreen (NEN Life Science Products)membrane in 10×SSC. Membrane was UV crosslinked twice (120 mJ) and bakedin a vacuum for 2 h at 80° C. Membrane was prehybridized at 42° C.overnight in hybridization solution [50% formamide, 5× Denhardt'ssolution, 1% SDS, 10% dextran sulfate, 1 mg sonicated salmon sperm DNA(boiled), 5× standard saline phosphate with EDTA (SSPE)]. Probes werelabeled by nick translation (Rigby et al., 1977). A fragment of CRG-L2(nucleotides 188-1243 of SEQ ID NO:1) was released with EcoRI from thepCR-TOPO4 vector. ³²P-labeled probe was added to the hybridizationbuffer and hybridized overnight at 42° C. Blots were washed-at RT in2×SSPE for 30 min and at 65° C. for 45 min in 2×SSPE, 2% SDS. Signalswere visualized by autoradiography or phosphoimagery.

[0042] For analysis of CRG-L2 in human tissue, RT-PCR was performed for25 cycles with primers hCRGL2a [5′-CATGGCAAGAACAGACTGGG-3′] (SEQ IDNO:9) and hCRGL2b [5′-GCCAGGAAACATCCCAAACTC-3′] (SEQ ID NO:10) and 10 μLof the reaction was electrophoresed on a 1% agarose/EtBr gel. The gelswere soaked in 1×TAE for 5 min, denatured for 30 min [1.5M NaCl, 0.5MNaOH], and neutralized for 30 min [1.5M NaCl, 0.5M Tris (pH 7.2), 1 mMEDTA (pH 8.0)]. DNA was transferred to a Hybond N membrane (Amersham)with 20×SSPE overnight. The membrane was baked for 30 min at 80° C. in avacuum oven and UV crosslinked twice (120 mJ). The membrane wasprehybridized at 42° C. for 3 h in hybridization solution [50%formamide, 5% Denhardt's, 3.4×SSPE, 10% dextran sulfate, 5% SDS, 1%sarkosyl, 100 mg sonicated salmon sperm DNA (SSS), 100 mg boiled SSS].Probes were labeled by nick translation (CRG-L2 fragment, nucleotides188-1243 of SEQ ID NO:1) and added to the hybridization solution.Membranes were hybridized overnight at 42° C. and were washed for 20 minat RT in 2×SSPE, 01% SDS and for 2 h at 65° C. in 0.5×SSPE, 0.2% SDS.Signals were visualized by autoradiography and phosphoimagery. Allprimers used in this study were synthesized at the University ofWisconsin-Madison Biotechnology Center.

[0043] In Situ Hybridization. In situ hybridization was performed asdescribed previously (Micales & Lyons, 2001, incorporated by referencein its entirety) with the CRG-L2 plasmid 5-2 (containing nucleotides82-1243 of SEQ ID NO:1) and AFP plasmid (containing nucleotides 726-1401of the AFP mRNA) in the plasmid pCR4-TOPO (Invitrogen). Sense andantisense probes were synthesized using T7 or SP6 with a MAXIscript kit(Ambion) to generate ³⁵S uridine triphosphate (UTP)-labeled riboprobes.Hybridized sections were exposed to emulsion (NTB-2; Eastman Kodak) inthe dark for 2 weeks before developing. After they were developed, thesections were counterstained with hematoxylin, mounted and viewed underboth light-field and dark-field illumination.

[0044] Multiple Tissue cDNA Panel. The mouse and human tissue cDNApanels (Clontech) were screened following manufacturer's instructions.After 28 cycles, 5 μl aliquots were removed at various timepoints. Themouse panel was screened with primers, GSP-970 and GSP-1241 (see RACEsection for primer sequences), and the human panel was screened withprimers, hCRGL2-C [5′-AGGGCCCACCAGGGCAGAAG-3′] (SEQ ID NO:11) andhCRGL2D [5′-ACATGCTTGGCTGCCGAGGG-3′] (SEQ ID NO:12).

[0045] Human Tissue. Human tissue and serum was procured from theUniversity of Wisconsin Surgical Pathology department, National DiseaseResearch Interchange, and the NCI Cooperative Human Tissue Network. Allsamples analyzed were primary tissues. As required by our IRB protocol,the identity of the patients was unknown. The excess tissue was frozenafter surgery and stored at −70° C.

Results

[0046] Cloning of CRG-L2 using Rapid Amplification of cDNA Ends. Byrepresentational difference analysis, a 282 bp fragment of anuncharacterized mRNA was isolated (Graveel et al., 2001). Using RT-PCRanalysis with primers located in the RDA fragment, this mRNA showedelevated expression in mouse liver tumors as compared to quiescent,regenerating, or newborn livers (FIG. 1a). The low level of expressionin the regenerating livers suggested the possibility that the increasedexpression was tumor-specific and would not occur in non-tumorigenicproliferative states of human liver, such as cirrhosis or hepatitis. Thecomplete cDNA was obtained via Rapid Amplification of cDNA Ends (RACE).Products from both 5′ and 3′ RACE were subcloned and sequenced.Sequencing the 3′ RACE products revealed three fragments which wereidentical at their 5′ ends due to the fixed location of thegene-specific primer. However, these fragments differed at their 3′ends, with the longer fragments containing, but extending past, thesequence of the shorter fragments. Each fragment contained a polyA tailat its 3′ end, indicating that there are multiple polyadenylation sites.The 5′ RACE products were all identical. By conceptually combining the5′ and 3′ RACE products, three mRNAs were identified that containedalternative 3′UTRs (FIG. 1b). The putative start codon is at nucleotide25 and the putative stop codon at nucleotide 1344 (see SEQ ID NO:1).Because it was known that this mRNA was upregulated in murine livertumors yet the function was unknown, this novel mRNA was named CancerRelated Gene-Liver 2 (CRG-L2).

[0047] To confirm the presence of all three of the murine CRG-L2 mRNAsand to determine which mRNA is predominantly expressed, a Northern blothybridization was performed using mRNA from quiescent livers and fourindividual liver tumors. A 1 kb fragment of the CRG-L2 open readingframe was used as a probe and four mRNAs were observed (FIG. 1c). The2.4, 3.0, and 5.5 kb mRNAs (designated as A, B, and C respectively inFIG. 1c) correspond to the 1967, 2380, and 4365 bp cloned cDNAs. Thesize of the observed mRNAs was longer than the RACE cDNA products due tothe polyA tails. A fourth mRNA (designated as D) of approximately 10 kbwas faintly detected but was not cloned via RACE presumably due to itslength. As expected based on previous RT-PCR results, none of the mRNAswere observed in the quiescent livers. The 5.5 kb mRNA was thepredominant form in the liver tumors and thus the sequence of the 4365nt mRNA has been deposited in Genbank as CRG-L2 (AF548022, SEQ ID NO:1).

[0048] To determine the structure of the CRG-L2 gene, the sequence ofthe mRNA was aligned to mouse chromosome 9 (31 cM) using the JacksonLaboratory and Ensembl Mouse Genome browsers (FIG. 1d). The CRG-L2 geneis comprised of 10 exons and nine introns that cover a minimum of 59 kb.An exact measure of the CRG-L2 gene is not yet possible because thereare gaps between the contigs that contain the introns between exons 1and 2 and exons 8 and 9.

[0049] The amino acid sequence (SEQ ID NO:2) of the 47.5 kDa CRG-L2protein was analyzed by the SMART analysis program (FIG. 2a) and wasfound to contain two collagen domains near the amino terminus (aminoacids 29-88 and 89-149 of SEQ ID NO:2) and a large olfactomedin domainwithin the C terminus (amino acid 189-433 of SEQ ID NO:2).Hydrophobicity analysis of the CRG-L2 protein revealed hydrophobicsequences within the first thirty amino acids of the amino terminus,which represent a leader sequence, suggesting that CRG-L2 is secreted(FIG. 2b). A serine was also present at amino acid 21 which isanticipated to be the cleavage site of the leader sequence. Regions ofhigh hydrophobicity were also present in the carboxy terminal region,which is anticipated to represent transmembrane domains.

[0050] The human sequence for CRG-L2 was pieced together by using theUCSC Human Genome Working Draft (http://genome.ucsc.edu/) to align thesequences. The resulting cDNA sequence is presented as SEQ ID NO:3 andthe putative amino acid sequence is presented as SEQ ID NO:4. Using theWilbur-Lipman DNA alignment method the mouse and human open readingframe (ORF) are found to be 82.4% identical. Using the Blosum62alignment method the mouse and human predicted protein products arefound to be 76% identical. Like the murine protein, the human proteincontains two putative collagen domains and one olfactomedin domain,located at amino acids 27-85, 86-145, and 177-395 of SEQ ID NO:4,respectively.

[0051] CRG-L2 is localized within chromosome 15q21.2 of the human genomeand a similar intron/exon structure is suggested by comparing the mousecDNA to the human genome. In the human genome data base at NCBI, cloneHs 15_(—)10351 (Genbank Accession No. NT_(—)010194), a contig from humanchromosome 15, has areas of significant homology to the mouse cDNAsequences. Because this region of the human genome has not been finishedin NCBI, the UCSC Human Genome Working Draft (http://genome.ucsc.edu/)was used to align the sequences in piecing together the human sequencefor CRG-L2. First, exons of the human CRG-L2 gene were identified byaligning the mouse CRG-L2 ORF to the human genome using the NBLASTprogram. Next, the identified exons were spliced together and putativeintrons were excised to form SEQ ID NO:3. SEQ ID NO:4 shows a predictedpolypeptide sequence encoded by SEQ ID NO:3. The skilled artisan willappreciate the possibility for some variation in the polynucleotide andpolypeptide sequences arising from uncertainty at putative splice sites.

[0052] CRG-L2 mRNA is upregulated in human hepatocellular carcinomas. Asnoted above, regions of human chromosome 15 are highly similar to mouseCRG-L2. Based on this similarity, primers were designed to detect humanCRG-L2 mRNA. Using these human primers, the level of expression ofCRG-L2 was measured in multiple human hepatocellular carcinomas and innormal livers. A combined method of RT-PCR and Southern blothybridization was used to measure the levels of human CRG-L2. CRG-L2mRNA was amplified by RT-PCR for 25 cycles and the PCR products weretransferred to a nylon membrane that was probed with a fragment of themurine CRG-L2 open reading frame (nucleotides 188-1243 of SEQ ID NO:1).As shown in FIG. 3, CRG-L2 mRNA is essentially undetectable in thenormal liver samples but can be detected in all five hepatocellularcarcinoma samples (middle panel). Extremely high expression is seen inHCC-2, as seen by the shorter exposure of the film (top panel). Accuratequantitation of the starting mRNA samples was verified by analysis ofGAPDH mRNA.

[0053] CRG-L2 is upregulated early in liver tumorigenesis. A veryimportant characteristic of a clinical marker for HCC would be earlyexpression during liver tumor development. Because it is difficult toobtain samples corresponding to early states of liver tumors from humancancer patients, we investigated the timing of expression of CRG-L2using the DEN-treated mouse model. After a single administration of DENto 12 day old mice, basophilic foci are visible by histological stainingat 12 weeks of age. Sequential development of hyperplastic nodules,hepatocellular adenomas, and hepatocellular carcinomas is observedbetween 12 weeks and 32 weeks of age in male mice (Moore et al., 1981;Vesselinovitch et al., 1985). Therefore, we sacrificed the DEN-treatedmice at 20 and 32 weeks of age. At 20 weeks of age, numerouspreneoplastic lesions were visible throughout the liver and by 32 weeksthe foci had progressed into hepatocellular adenomas/carcinomas (Haniganet al., 1988). Paraformaldehyde fixed sections from 20 and 32 weeklivers were probed with either an antisense (to detect CRG-L2 mRNA) orsense (negative control) CRG-L2 probe.

[0054] We began by analysis of the 32 week tumors because our RT-PCRresults clearly showed that CRG-L2 is upregulated at this stage.Although we expected to detect CRG-L2 mRNA in the 32 week tumors, insitu hybridization can provide additional information that cannot beobtained by RT-PCR analysis. For example, tumor-specific genes maydemonstrate a constant level of expression throughout a tumor or theexpression can be localized to specific cell types or spatial locations(e.g. the periphery of the tumor). Using in situ hybridization, weobserved that CRG-L2 mRNA was upregulated in hepatocytes throughout theentire tumor. We note that CRG-L2 was detected in only 69% (311/453) ofthe tumors examined using in situ hybridization but was detected in allseven tumors examined by RT-PCR (FIG. 1a). This could be due to the factthat only seven tumors were analyzed in FIG. 1a or because RT-PCR ismore sensitive than in situ hybridization.

[0055] To determine if CRG-L2 is upregulated at early stages ofhepatocarcinogenesis, the expression of CRG-L2 was examined in thepreneoplastic foci using in situ hybridization. Interestingly, we foundthat CRG-L2 mRNA can be detected in preneoplastic foci. The pattern ofCRG-L2 expression appears to be consistent throughout the focus with nolocalization within any individual region. We found that CRG-L2 ishighly upregulated in 55% of the foci (220/403) but that there is noobvious histological differences in those foci which do or do notexpress CRG-L2; e.g. CRG-L2 is upregulated in both basophilic andeosinophilic foci and in foci with extensive fat or collagen deposits.AFP was found to be upregulated in 30% of preneoplastic foci (92/304)although the expression pattern was often restricted to various regionsof the focus and not as uniformly distributed as CRG-L2. In otherstudies, AFP has been shown to be expressed in only 23% of 28 week oldDEN-treated B6C3F1 mice (Koen et al., 1983) and 24% of humanhepatocellular carcinomas by immunohistochemistry (Borscheri et al.,2001). In comparison to AFP in these studies, CRG-L2 may be a moresensitive marker for the detection of early HCC.

[0056] CRG-L2 displays restricted expression in normal tissues. Acharacteristic of a good clinical marker for HCC is tumor-specificexpression; i.e. low expression in all normal tissues not just in thetissue from which the tumor is derived. Although CRG-L2 mRNA was notdetected in normal mouse liver, it was possible that the mRNA wasexpressed in other normal tissues. The expression of CRG-L2 was examinedin mouse and human tissues using a multiple tissue cDNA panel. Becauseperforming high numbers of PCR cycles can sometimes obscure differentialexpression, aliquots of the PCR products were taken out after variouscycles (30-34). We found that CRG-L2 is primarily expressed in the mousetestis with moderate expression in skeletal muscle (FIG. 4a). In humantissues, CRG-L2 was expressed primarily in the placenta (FIG. 4b). Thepattern of CRG-L2 expression, high in tumors, but normally expressed intestis and placenta, resembles expression patterns of genes known ascancer-testis antigens (CT antigen). Cancer-testis antigens are a groupof genes classified by their exclusive expression in the testis andother reproductive tissues and diverse human cancers. The above findingssuggest that CRG-L2 is a potential CT antigen.

[0057] CRG-L2 is a CT antigen. The examination of CRG-L2 expressionrevealed that CRG-L2 mRNA is expressed at very low levels in all normaltissues except in the mouse testis and human placenta. Therefore, CRG-L2falls into a class of genes designated as cancer-testis antigens (CTantigen). The characteristics of CT antigens are a lack of expression innormal tissues, except reproductive tissues, and high levels ofexpression in a wide range of tumor types. Currently there are more thanten genes identified that are CT antigens, one of which, PAGE, alsoshows high expression levels in the placenta (Brinkman et al., 1998).Most CT antigens map to the X chromosome, but SCP-1 (Türeci et al.,1998), CT9 (Scanlan et al., 2000), and OY-TES-1 (Ono et al., 2001) mapto other chromosomes, as does CRG-L2. CT antigens are intriguingtherapeutic targets for immunotherapy because of their limitedexpression in normal tissues and the fact that the testis and placentaare immune-privileged sites. However, the biological function and therelationship to malignancy of most of these genes is unknown (Ono etal., 2001; Scanlan et al., 2002). With regard to CRG-L2, the proteinstructure indicates that CRG-L2 belongs to a family ofolfactomedin-related proteins, which includes olfactomedin,myocilin/TIGR, noelin-1, and hGC-1. Olfactomedin-related genes havecharacteristic tissue-restricted expression patterns suggesting aspecialized function for each protein (Richards et al., 1998; Zhang etal., 2002). Based on tissue localization of several olfactomedin familymembers and the function of TIGR/myocilin, it is possible thatolfactomedin-related proteins play an important role in protein-proteininteractions within the extracellular matrix (Kulkarni et al., 2000).CRG-L2 also contains two collagen domains; proteins that containcollagen domains are also often involved in the structure of theextracellular matrix.

[0058] Our results clearly indicate that expression of CRG-L2 isincreased in tumors. This increased expression in tumors and restrictedpattern of expression in normal tissues indicates that CRG-L2 is atumor-specific antigen. It is thus anticipated that a patient willdisplay an immunogenic response to CRG-L2, making CRG-L2 a marker thatcan be detected using blood samples to allow more cost-effectivescreening of a larger number of high risk patients.

[0059] The polynucleotide and polypeptide sequences disclosed hereinprovide a skilled artisan with the ability to assess using conventionalmethods the expression levels of the human CRG-L2 gene in an array oftissues and more specifically to monitor the expression of the gene inhuman liver regions suspected of liver cancer or preneoplasticdevelopment as compared to normal human liver tissue. Likewise,antibodies directed to a portion of the human protein can be producedand used as diagnostic agents for assessing protein levels in varioushuman tissues including liver tumors. In addition, over-expressed CRG-L2from liver tumor cells and preneoplastic liver cells is expected to besecreted into the blood and the blood level of this protein can beeasily monitored by various methods known to one of ordinary skill inthe art. A patient having liver cancer and preneoplastic development inthe liver may also develop an immune response to CRG-L2 and thus anantibody to CRG-L2 may be detected in the blood of the patient.

[0060] The present invention is not intended to be limited to theforegoing example, but rather to encompass all such variations andmodifications as come within the scope of the appended claims.

References

[0061] (All references listed below are herein incorporated by referencein their entirety)

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1 12 1 4365 DNA mouse CDS (25)..(1341) variation (1937) From thisposition forward, first variant 3′ UTR sequence readsaacccattaaaaaaaaaaaaaaaaaaaagt 1 cacatccgtg cggagagcca ggac atg atg atgatg atg acc tac tcc atg 51 Met Met Met Met Met Thr Tyr Ser Met 1 5 gtgccg att cga gtg atg ata gac ctg tgc aac agt acc cag ggc atc 99 Val ProIle Arg Val Met Ile Asp Leu Cys Asn Ser Thr Gln Gly Ile 10 15 20 25 tgcctc aca gga cca ccg ggc cca cca gga cct cca gga gcc ggc ggg 147 Cys LeuThr Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Ala Gly Gly 30 35 40 tta ccaggc cac aat gga tca gat gga cag cct ggt ctc cag ggc cca 195 Leu Pro GlyHis Asn Gly Ser Asp Gly Gln Pro Gly Leu Gln Gly Pro 45 50 55 aaa gga gaaaaa gga gca att ggc aag aga gga aaa atg ggg tta cct 243 Lys Gly Glu LysGly Ala Ile Gly Lys Arg Gly Lys Met Gly Leu Pro 60 65 70 gga gcc acc ggaaat cca ggg gaa aag gga gaa aag gga gat gct ggt 291 Gly Ala Thr Gly AsnPro Gly Glu Lys Gly Glu Lys Gly Asp Ala Gly 75 80 85 gaa ctg ggt cta cctgga aat gag ggc cca cca ggg cag aaa ggt gac 339 Glu Leu Gly Leu Pro GlyAsn Glu Gly Pro Pro Gly Gln Lys Gly Asp 90 95 100 105 aag gga gac aaagga gac gtg tcc aat gac gtg ctt ttg aca ggt gcc 387 Lys Gly Asp Lys GlyAsp Val Ser Asn Asp Val Leu Leu Thr Gly Ala 110 115 120 aaa ggt gac caaggt ccc cct ggc ccc cct gga cct cca ggg cct cca 435 Lys Gly Asp Gln GlyPro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro 125 130 135 ggc cct cct ggaagc aga aga tcc aaa ggc cct cgg cca cca aac gtg 483 Gly Pro Pro Gly SerArg Arg Ser Lys Gly Pro Arg Pro Pro Asn Val 140 145 150 ttc aac agc cagtgt cca ggg gag acg tgt gtc ata ccc aat gat gat 531 Phe Asn Ser Gln CysPro Gly Glu Thr Cys Val Ile Pro Asn Asp Asp 155 160 165 acc ttg gtg ggaaga gct gat gag aaa gca aat gaa cgc cat tca cca 579 Thr Leu Val Gly ArgAla Asp Glu Lys Ala Asn Glu Arg His Ser Pro 170 175 180 185 caa aca gaatct atg atc act tcc att ggc aac cca gcc caa gtc cta 627 Gln Thr Glu SerMet Ile Thr Ser Ile Gly Asn Pro Ala Gln Val Leu 190 195 200 aaa gtg agagag act ttt ggg act tgg atg aga gag tct gct aac aaa 675 Lys Val Arg GluThr Phe Gly Thr Trp Met Arg Glu Ser Ala Asn Lys 205 210 215 agt gac gaccgc att tgg gtg act gaa cat ttt tca ggc atc atg gtg 723 Ser Asp Asp ArgIle Trp Val Thr Glu His Phe Ser Gly Ile Met Val 220 225 230 aag gag ttcaaa gac ctg ccg gcg ctc ctc aat agc agc ttc acc ctc 771 Lys Glu Phe LysAsp Leu Pro Ala Leu Leu Asn Ser Ser Phe Thr Leu 235 240 245 ctc cac ctccca cat tat ttc cac ggc tgt ggg cac gct gtt tac aac 819 Leu His Leu ProHis Tyr Phe His Gly Cys Gly His Ala Val Tyr Asn 250 255 260 265 aac tctctc tac tac cac aaa gga ggc tcc aac acc ata gtg aga ttt 867 Asn Ser LeuTyr Tyr His Lys Gly Gly Ser Asn Thr Ile Val Arg Phe 270 275 280 gaa tttggg aaa gag aca cct cag act ctg aag ctg gaa aat gct ttg 915 Glu Phe GlyLys Glu Thr Pro Gln Thr Leu Lys Leu Glu Asn Ala Leu 285 290 295 tat tttgat cga aaa tac ctc ttt gca aat tcc aag act tac ttc aac 963 Tyr Phe AspArg Lys Tyr Leu Phe Ala Asn Ser Lys Thr Tyr Phe Asn 300 305 310 ata gcagtg gat gag aag ggc atc tgg att atc tac gct tca agt gtg 1011 Ile Ala ValAsp Glu Lys Gly Ile Trp Ile Ile Tyr Ala Ser Ser Val 315 320 325 gat ggctca agc atc ctt gta gca cag ctg gat ggg agg aca ttc tcc 1059 Asp Gly SerSer Ile Leu Val Ala Gln Leu Asp Gly Arg Thr Phe Ser 330 335 340 345 gtgaca cag cac atc aac acc aca tac ccc aaa tcc aag gct ggc aat 1107 Val ThrGln His Ile Asn Thr Thr Tyr Pro Lys Ser Lys Ala Gly Asn 350 355 360 gccttc ata gcc cga ggg atc ctc tat gtc aca gac acc aaa gat acg 1155 Ala PheIle Ala Arg Gly Ile Leu Tyr Val Thr Asp Thr Lys Asp Thr 365 370 375 agggtc acg ttt gcc ttt gat ttg tta gga gga aag caa atc aat gca 1203 Arg ValThr Phe Ala Phe Asp Leu Leu Gly Gly Lys Gln Ile Asn Ala 380 385 390 aacttt gat ttc aga atg tcc cag tct gtt ctt gcc atg ctg tca tac 1251 Asn PheAsp Phe Arg Met Ser Gln Ser Val Leu Ala Met Leu Ser Tyr 395 400 405 aacatg aga gat cag cat tta tac tcg tgg gaa gat ggc cat ctg atg 1299 Asn MetArg Asp Gln His Leu Tyr Ser Trp Glu Asp Gly His Leu Met 410 415 420 425ctc tat cct gtg cag ttt ctg tca gcg gca tca agt cag cgg 1341 Leu Tyr ProVal Gln Phe Leu Ser Ala Ala Ser Ser Gln Arg 430 435 tagggttccctcggctgtct gctccctctc tatactccac attgtctagg gtttggtcaa 1401 gcccaacagaaagctagccg gtaaaggata cccaggcact cggagcgtaa gcccatgcca 1461 cgggctcttgcacaagcggc gagtccgctc taagccaggt tgttgaaata gctacagatt 1521 agaaatggatgtggaagaga tctggtgacc cagtatccct cctcaaactc agcaagttag 1581 ctctcccccgaccgtagcgt ccccataggt aatacgaaac atctgagtat gactgacatt 1641 tcctcttcctagatgaaatt ctgtgattct tgcctgatta tatattagaa tgctttctgg 1701 attcttttttttttttttct ccacatgtaa gtgagcttac ttgcagcttg aggggtgggc 1761 ctttcagtgatgacttattt ggtatttagg gaaggtgcac tggctcttat ggcttctaag 1821 gttttattttattcataatt tgttattttc tctgaatatt cacctaccac tacagaatga 1881 tcattgttttcagctcctaa acacaaatcc aagattaata aacaaacaaa caaaccatga 1941 atagatacaggctcagaact ctaaatggag ctgcatcagg cccataggcc atctagatgc 2001 tgcaatttctgatcatattg tttgctgctg ggaaagtaaa caggatatct tcagttcgtg 2061 gtcccttttgccaaggccat gggattgtta tcagagtgtc aaacactaag tggccaataa 2121 tctggttagaagcatggaaa catgatggtt ttttcagaaa acaggcacca tttatactta 2181 ctgtttagaatgagggaagg caattggctc aaaggccaaa gtcagcttag ctctttttcc 2241 tgtaccatcgcatccctgca cctaagaatc tcgcctcaga gtgtgtcagc agtgaagcag 2301 agccgctctgtaaatcctga accattactg cctggccttt acagaaagaa agaaaaaaaa 2361 atgttgacctttcatctaag gacagggaac gagccaggtt ctcagaaggg ctcactccct 2421 gagtctggttaggctttcta cggactgaca ggcagcattt tatgtggctt gggctttggc 2481 agagggaacagctaaggaca gcatcagatg gagtaagaga acctccagcc gtggagatgt 2541 tcactcccacgtggtcctca aagttgggtc tgtcctcttg gatagcaagg atctagttta 2601 attggttcctacaagacctt aaataaccac gttctctgtc aactcattga gttccaggca 2661 ggcctgtggagcttcaaaga ggaagctgtg gatttcatcg cccccccccc ccggaatata 2721 gaaaaagacactacagaaac tgtccaggaa agactggcca gctgttccaa acccactctc 2781 agtgggcctgtgacctggtt tagttttttt aatagaagca tcttgaggct tggggtatgc 2841 attttaactatttaactttc cctgccctct gaaagcaccc aggcagctgt tactggtgaa 2901 cctgttgagttctcaaggtc atgggtccca aagcttcccc acttcttgat tagatggttt 2961 tgcagttggtcatcacagct tttaaagata ttctctcaga ttcatttgtt gcaatgtaga 3021 gttctaatgttttatcagtg tatctaatga atggtattgt tcttttaaag tatttaaata 3081 tgagatactgtttctgagtg cggtagacct ggatatacat ataattccat ttttttatta 3141 tttagtagcattgctgagaa tagatacaat actaattgta catacaagca aaatagttta 3201 gttattgaattagctcattt ttaatatctg aactagcaaa tgtcttagct ttcctttact 3261 tttttctttcttttcctttc ttttctcttc ttttcctttc tttcctttcc ttttcttttc 3321 ttttttttaaagcaatgtct ttgtgttcgc ccagacttat cacaaactcc tgcttcagat 3381 tcctggggctgggaccacag gcacagtggc tctttgactc tcttaattgt gtgtaaggaa 3441 tcatacatatactcacgatt agagaaactc gtctgaagat tttgtttctt ttcatggttg 3501 tttctttctttctttctttc tttctttctt tcgttatagt gtagtgggat tagaacaagt 3561 aaggttgactggtgtttaat gaatttatct ttgcagaagg aaaggaatta aggttttatt 3621 ccttttcttgcaaacaggac ttcattctat atcactcaac acagtgtttc aggctcactg 3681 ctaaaatagtgtgcacatct tatattttta aatgaagata gtaatcaacc ctgctgtcac 3741 ttgtagccaagctgttctaa aagcacttca tttatgtctg tatgaaatca agtgattctc 3801 caattcctctgaaatctaaa gtagatacca ttatactaga aaccacacct tccagcttca 3861 aaggtaggccagactcaaca tttacaaagc atttctatta actaatatag agtccaacta 3921 aggttgcagagttggctctg gcctcaatgt atcatgtatc aatgtatcag agaacgtggt 3981 ccgggctgaatatttcagat caattctggt gctgggctca ttcgaagtct ttttaccctc 4041 ataatcaaatgacaaggtga gatgacaaat gaggaagcac agtccttgaa aagtcactcg 4101 tcatcctccaagcatagcaa gtaccttact caggcattgc ctgtctggtg ttgagctacc 4161 tgaaggaaaagtggggggtg gagctcttca gttttcatca gtgctgtggc cttatttatc 4221 tcataatctcccatcagtaa ccacagattc taaacgacca gcaagtaaca gttgtaagta 4281 gtaaaataaaattatcctga atataatcac aaaaaaaaaa aaaaaaaaaa aagaaaaaaa 4341 aaaaaaaaaaaaaaaaaaaa aagt 4365 2 439 PRT mouse 2 Met Met Met Met Met Thr Tyr SerMet Val Pro Ile Arg Val Met Ile 1 5 10 15 Asp Leu Cys Asn Ser Thr GlnGly Ile Cys Leu Thr Gly Pro Pro Gly 20 25 30 Pro Pro Gly Pro Pro Gly AlaGly Gly Leu Pro Gly His Asn Gly Ser 35 40 45 Asp Gly Gln Pro Gly Leu GlnGly Pro Lys Gly Glu Lys Gly Ala Ile 50 55 60 Gly Lys Arg Gly Lys Met GlyLeu Pro Gly Ala Thr Gly Asn Pro Gly 65 70 75 80 Glu Lys Gly Glu Lys GlyAsp Ala Gly Glu Leu Gly Leu Pro Gly Asn 85 90 95 Glu Gly Pro Pro Gly GlnLys Gly Asp Lys Gly Asp Lys Gly Asp Val 100 105 110 Ser Asn Asp Val LeuLeu Thr Gly Ala Lys Gly Asp Gln Gly Pro Pro 115 120 125 Gly Pro Pro GlyPro Pro Gly Pro Pro Gly Pro Pro Gly Ser Arg Arg 130 135 140 Ser Lys GlyPro Arg Pro Pro Asn Val Phe Asn Ser Gln Cys Pro Gly 145 150 155 160 GluThr Cys Val Ile Pro Asn Asp Asp Thr Leu Val Gly Arg Ala Asp 165 170 175Glu Lys Ala Asn Glu Arg His Ser Pro Gln Thr Glu Ser Met Ile Thr 180 185190 Ser Ile Gly Asn Pro Ala Gln Val Leu Lys Val Arg Glu Thr Phe Gly 195200 205 Thr Trp Met Arg Glu Ser Ala Asn Lys Ser Asp Asp Arg Ile Trp Val210 215 220 Thr Glu His Phe Ser Gly Ile Met Val Lys Glu Phe Lys Asp LeuPro 225 230 235 240 Ala Leu Leu Asn Ser Ser Phe Thr Leu Leu His Leu ProHis Tyr Phe 245 250 255 His Gly Cys Gly His Ala Val Tyr Asn Asn Ser LeuTyr Tyr His Lys 260 265 270 Gly Gly Ser Asn Thr Ile Val Arg Phe Glu PheGly Lys Glu Thr Pro 275 280 285 Gln Thr Leu Lys Leu Glu Asn Ala Leu TyrPhe Asp Arg Lys Tyr Leu 290 295 300 Phe Ala Asn Ser Lys Thr Tyr Phe AsnIle Ala Val Asp Glu Lys Gly 305 310 315 320 Ile Trp Ile Ile Tyr Ala SerSer Val Asp Gly Ser Ser Ile Leu Val 325 330 335 Ala Gln Leu Asp Gly ArgThr Phe Ser Val Thr Gln His Ile Asn Thr 340 345 350 Thr Tyr Pro Lys SerLys Ala Gly Asn Ala Phe Ile Ala Arg Gly Ile 355 360 365 Leu Tyr Val ThrAsp Thr Lys Asp Thr Arg Val Thr Phe Ala Phe Asp 370 375 380 Leu Leu GlyGly Lys Gln Ile Asn Ala Asn Phe Asp Phe Arg Met Ser 385 390 395 400 GlnSer Val Leu Ala Met Leu Ser Tyr Asn Met Arg Asp Gln His Leu 405 410 415Tyr Ser Trp Glu Asp Gly His Leu Met Leu Tyr Pro Val Gln Phe Leu 420 425430 Ser Ala Ala Ser Ser Gln Arg 435 3 1203 DNA human CDS (1)..(1200) 3atg ctg atg atg atg acc tac tcc atg gtg ccg atc cga gtg atg gtg 48 MetLeu Met Met Met Thr Tyr Ser Met Val Pro Ile Arg Val Met Val 1 5 10 15gac ctg tgc aac agc acc aag ggc atc tgc ctc aca gga cct ccg gga 96 AspLeu Cys Asn Ser Thr Lys Gly Ile Cys Leu Thr Gly Pro Pro Gly 20 25 30 gccggc ggg ttg cca gga cac aac gga ttg gat gga cag cct ggt cct 144 Ala GlyGly Leu Pro Gly His Asn Gly Leu Asp Gly Gln Pro Gly Pro 35 40 45 cag ggccca aaa gga gaa aaa gga gca aat gga aaa aga gga aaa atg 192 Gln Gly ProLys Gly Glu Lys Gly Ala Asn Gly Lys Arg Gly Lys Met 50 55 60 ggg ata cctgga gct gca gga aat cca ggg gaa agg gga gaa aag gga 240 Gly Ile Pro GlyAla Ala Gly Asn Pro Gly Glu Arg Gly Glu Lys Gly 65 70 75 80 gac cat ggtgaa ctg ggc ctg cag gga aat gag ggc cca cca ggg cag 288 Asp His Gly GluLeu Gly Leu Gln Gly Asn Glu Gly Pro Pro Gly Gln 85 90 95 aag gga gaa aagggt gac aaa gga gat gtg tcc aac gac gtg ctc ctg 336 Lys Gly Glu Lys GlyAsp Lys Gly Asp Val Ser Asn Asp Val Leu Leu 100 105 110 ggt gcc aaa ggtgac caa ggc cca ccc ggt cca cct ggg ccc cca ggc 384 Gly Ala Lys Gly AspGln Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly 115 120 125 cct cca ggt cctcca ggg ccc cct gga agc aga aga gcc aaa ggc cct 432 Pro Pro Gly Pro ProGly Pro Pro Gly Ser Arg Arg Ala Lys Gly Pro 130 135 140 cgg cag cca agcatg ttc aac ggc cag tgc cca ggt gag act tgt gcc 480 Arg Gln Pro Ser MetPhe Asn Gly Gln Cys Pro Gly Glu Thr Cys Ala 145 150 155 160 ata cca aatgat gat acc ttg gtt gga aaa gct gat gag aaa gcc aaa 528 Ile Pro Asn AspAsp Thr Leu Val Gly Lys Ala Asp Glu Lys Ala Lys 165 170 175 tcc atg atcact tcc att gga aac cca gtg caa gta ctg aaa gtg aca 576 Ser Met Ile ThrSer Ile Gly Asn Pro Val Gln Val Leu Lys Val Thr 180 185 190 gag aca tttggg act tgg ata aga gag tct gct aac aag agt gat gac 624 Glu Thr Phe GlyThr Trp Ile Arg Glu Ser Ala Asn Lys Ser Asp Asp 195 200 205 cgg att tgggtg aca gag cat ttt tca ggt cca cct tcc ata cta ttt 672 Arg Ile Trp ValThr Glu His Phe Ser Gly Pro Pro Ser Ile Leu Phe 210 215 220 cca tgg ctgtgg gca cgt tgt tta caa caa ctc tct cta cta cca caa 720 Pro Trp Leu TrpAla Arg Cys Leu Gln Gln Leu Ser Leu Leu Pro Gln 225 230 235 240 agg ggattt gaa ttt ggc cag gaa aca tcc caa act ctg aag ctt gaa 768 Arg Gly PheGlu Phe Gly Gln Glu Thr Ser Gln Thr Leu Lys Leu Glu 245 250 255 aat gccttg tat ttt gat cga aaa tac ctt ttt gca aat tcc aaa act 816 Asn Ala LeuTyr Phe Asp Arg Lys Tyr Leu Phe Ala Asn Ser Lys Thr 260 265 270 tac ttcaat cta gct gta gat gaa aag ggc ctt tgg att atc tat gcg 864 Tyr Phe AsnLeu Ala Val Asp Glu Lys Gly Leu Trp Ile Ile Tyr Ala 275 280 285 tca agtgtg gac ggc tcg agc att ctt gta gca caa ctg gat gag agg 912 Ser Ser ValAsp Gly Ser Ser Ile Leu Val Ala Gln Leu Asp Glu Arg 290 295 300 aca ttctca gtg gtg caa cac gtc aat acc acg tac cct aaa tcc aag 960 Thr Phe SerVal Val Gln His Val Asn Thr Thr Tyr Pro Lys Ser Lys 305 310 315 320 gctggc aac gcc ttc att gcc cga gga atc ctc tat gtc aca gac acc 1008 Ala GlyAsn Ala Phe Ile Ala Arg Gly Ile Leu Tyr Val Thr Asp Thr 325 330 335 aaagat atg agg gtc aca ttt gcc ttt gat ttg tta gga ggg aaa cag 1056 Lys AspMet Arg Val Thr Phe Ala Phe Asp Leu Leu Gly Gly Lys Gln 340 345 350 atcaat gca aac ttt gat tta aga act tcc cag tct gtt ctt gcc atg 1104 Ile AsnAla Asn Phe Asp Leu Arg Thr Ser Gln Ser Val Leu Ala Met 355 360 365 ttagca tac aac atg aga gat cag cat tta tat tca tgg gaa gat ggc 1152 Leu AlaTyr Asn Met Arg Asp Gln His Leu Tyr Ser Trp Glu Asp Gly 370 375 380 cattta atg ctt tat cct gtg cag ttt ttg tca act acc tta aat cag 1200 His LeuMet Leu Tyr Pro Val Gln Phe Leu Ser Thr Thr Leu Asn Gln 385 390 395 400tga 1203 4 400 PRT human 4 Met Leu Met Met Met Thr Tyr Ser Met Val ProIle Arg Val Met Val 1 5 10 15 Asp Leu Cys Asn Ser Thr Lys Gly Ile CysLeu Thr Gly Pro Pro Gly 20 25 30 Ala Gly Gly Leu Pro Gly His Asn Gly LeuAsp Gly Gln Pro Gly Pro 35 40 45 Gln Gly Pro Lys Gly Glu Lys Gly Ala AsnGly Lys Arg Gly Lys Met 50 55 60 Gly Ile Pro Gly Ala Ala Gly Asn Pro GlyGlu Arg Gly Glu Lys Gly 65 70 75 80 Asp His Gly Glu Leu Gly Leu Gln GlyAsn Glu Gly Pro Pro Gly Gln 85 90 95 Lys Gly Glu Lys Gly Asp Lys Gly AspVal Ser Asn Asp Val Leu Leu 100 105 110 Gly Ala Lys Gly Asp Gln Gly ProPro Gly Pro Pro Gly Pro Pro Gly 115 120 125 Pro Pro Gly Pro Pro Gly ProPro Gly Ser Arg Arg Ala Lys Gly Pro 130 135 140 Arg Gln Pro Ser Met PheAsn Gly Gln Cys Pro Gly Glu Thr Cys Ala 145 150 155 160 Ile Pro Asn AspAsp Thr Leu Val Gly Lys Ala Asp Glu Lys Ala Lys 165 170 175 Ser Met IleThr Ser Ile Gly Asn Pro Val Gln Val Leu Lys Val Thr 180 185 190 Glu ThrPhe Gly Thr Trp Ile Arg Glu Ser Ala Asn Lys Ser Asp Asp 195 200 205 ArgIle Trp Val Thr Glu His Phe Ser Gly Pro Pro Ser Ile Leu Phe 210 215 220Pro Trp Leu Trp Ala Arg Cys Leu Gln Gln Leu Ser Leu Leu Pro Gln 225 230235 240 Arg Gly Phe Glu Phe Gly Gln Glu Thr Ser Gln Thr Leu Lys Leu Glu245 250 255 Asn Ala Leu Tyr Phe Asp Arg Lys Tyr Leu Phe Ala Asn Ser LysThr 260 265 270 Tyr Phe Asn Leu Ala Val Asp Glu Lys Gly Leu Trp Ile IleTyr Ala 275 280 285 Ser Ser Val Asp Gly Ser Ser Ile Leu Val Ala Gln LeuAsp Glu Arg 290 295 300 Thr Phe Ser Val Val Gln His Val Asn Thr Thr TyrPro Lys Ser Lys 305 310 315 320 Ala Gly Asn Ala Phe Ile Ala Arg Gly IleLeu Tyr Val Thr Asp Thr 325 330 335 Lys Asp Met Arg Val Thr Phe Ala PheAsp Leu Leu Gly Gly Lys Gln 340 345 350 Ile Asn Ala Asn Phe Asp Leu ArgThr Ser Gln Ser Val Leu Ala Met 355 360 365 Leu Ala Tyr Asn Met Arg AspGln His Leu Tyr Ser Trp Glu Asp Gly 370 375 380 His Leu Met Leu Tyr ProVal Gln Phe Leu Ser Thr Thr Leu Asn Gln 385 390 395 400 5 20 DNAArtificial Sequence Description of Artificial SequencePCR primer 5gcatggcaag aacagactgg 20 6 20 DNA Artificial Sequence Description ofArtificial SequencePCR primer 6 ggatgagaag ggcatctgga 20 7 19 DNAArtificial Sequence Description of Artificial SequencePCR primer 7caacaacctg gcttagagc 19 8 19 DNA Artificial Sequence Description ofArtificial SequencePCR primer 8 gccatctgat gctctatcc 19 9 20 DNAArtificial Sequence Description of Artificial SequencePCR primer 9catggcaaga acagactggg 20 10 21 DNA Artificial Sequence Description ofArtificial SequencePCR primer 10 gccaggaaac atcccaaact c 21 11 20 DNAArtificial Sequence Description of Artificial SequencePCR primer 11agggcccacc agggcagaag 20 12 20 DNA Artificial Sequence Description ofArtificial SequencePCR primer 12 acatgcttgg ctgccgaggg 20

We claim:
 1. An isolated nucleic acid comprising a polynucleotideselected from the group consisting of (1) a first nucleotide sequencethat encodes a polypeptide selected from the group consisting of aminoacid 22 to amino acid 439 of SEQ ID NO:2, amino acid 22 to amino acid400 of SEQ ID NO:4, a sequence that is at least about 68% identical toamino acid 22 to amino acid 439 of SEQ ID NO:2, and a sequence that isat least about 68% identical to amino acid 22 to amino acid 400 of SEQID NO:4, (2) a second nucleotide sequence that is at least 80% identicalto the first nucleotide sequence, and (3) a complement of the first orsecond nucleotide sequence.
 2. The isolated nucleic acid of claim 1,wherein the nucleic acid consists of a polynucleotide selected from thegroup consisting of (1) a first nucleotide sequence that encodes apolypeptide selected from the group consisting of amino acid 22 to aminoacid 439 of SEQ ID NO:2, amino acid 22 to amino acid 400 of SEQ ID NO:4,a sequence that is at least about 68% identical to amino acid 22 toamino acid 439 of SEQ ID NO:2, and a sequence that is at least about 68%identical to amino acid 22 to amino acid 400 of SEQ ID NO:4, (2) asecond nucleotide sequence that is at least 80% identical to the firstnucleotide sequence, and (3) a complement of the first or secondnucleotide sequence.
 3. The isolated nucleic acid of claim 1, whereinthe nucleic acid comprises a polynucleotide selected from the groupconsisting of nucleotide 88 to nucleotide 1341 of SEQ ID NO:1,nucleotide 64 to nucleotide 1200 of SEQ ID NO:3, and a complement of anyof the foregoing.
 4. A genetic construct comprising a polynucleotide ofclaim 1 operably linked a heterologous transcriptional promoter.
 5. Ahost cell comprising the genetic construct of claim
 4. 6. The isolatednucleic acid of claim 1, wherein the nucleic acid comprises apolynucleotide selected from the group consisting of (1) a firstnucleotide sequence that encodes a polypeptide selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO:4, a sequence that is at leastabout 70% identical to SEQ ID NO:2, and a sequence that is at leastabout 70% identical to SEQ ID NO:4, (2) a second nucleotide sequencethat is at least 80% identical to the first nucleotide sequence, and (3)a complement of the first or second nucleotide sequence.
 7. The isolatednucleic acid of claim 6, wherein the nucleic acid consists of apolynucleotide selected from the group consisting of (1) a firstnucleotide sequence that encodes a polypeptide selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO:4, a sequence that is at least 70%identical to SEQ ID NO:2, and a sequence that is at least 70% identicalto SEQ ID NO:4, (2) a second nucleotide sequence that is at least 80%identical to the first nucleotide sequence, and (3) a complement of thefirst or second nucleotide sequence.
 8. The isolated nucleic acid ofclaim 6, wherein the nucleic acid comprises a polynucleotide selectedfrom the group consisting of nucleotide 25 to nucleotide 1341 of SEQ IDNO:1, nucleotide 1 to nucleotide 1200 of SEQ ID NO:3, and a complementof any of the foregoing.
 9. A genetic construct comprising apolynucleotide of claim 6 operably linked a heterologous transcriptionalpromoter.
 10. A host cell comprising the genetic construct of claim 9.11. An isolated polypeptide comprising an amino acid sequence encoded bythe first nucleotide sequence in claim
 1. 12. An isolated polypeptideconsisting of an amino acid sequence encoded by the first nucleotidesequence in claim
 1. 13. The isolated polypeptide of claim 11, whereinthe polypeptide comprises an amino acid sequence selected from the groupconsisting of amino acid 22 to amino acid 439 of SEQ ID NO:2 and aminoacid 22 to amino acid 400 of SEQ ID NO:4.
 14. An isolated polypeptidecomprising an amino acid sequence encoded by the first nucleotidesequence in claim
 6. 15. An isolated polypeptide consisting of an aminoacid sequence encoded by the first nucleotide sequence in claim
 6. 16.The isolated polypeptide of claim 14, wherein the polypeptide comprisesan amino acid sequence selected from the group consisting of SEQ ID NO:2and SEQ ID NO:4.
 17. An antibody that specifically binds to apolypeptide consisting of an amino acid sequence encoded by the firstnucleotide sequence in claim
 1. 18. An antibody that specifically bindsto a polypeptide consisting of an amino acid sequence encoded by thefirst nucleotide sequence in claim
 6. 19. A method for identifying anagent that can modulate the expression of a polynucleotide of claim 1,the method comprising the steps of: exposing a cell that comprises apolynucleotide of claim 1 under the control of its native promoter;measuring the expression of the polynucleotide in the cell; andcomparing the expression to that in a control cell that is not exposedto the test agent, wherein a higher or lower than the expression in thecontrol cell indicates that the agent can modulate the expression of thepolynucleotide.
 20. The method of claim 19, wherein the cell thatcomprises a polynucleotide of claim 1 under the control of its nativepromoter is a cell selected from the group consisting of a liver cell, abreast cell, a kidney cell and a colon cell.
 21. The method of claim 19,wherein the expression is measured at the mRNA level.
 22. The method ofclaim 19, wherein the expression is measured at the protein level.
 23. Amethod for diagnosing a cancer or preneoplastic development in a tissueor organ of a human or non-human animal, the method comprising the stepsof: measuring the expression of a polynucleotide of claim 1 in cells ofthe tissue or organ obtained from a region suspected of cancer orpreneoplastic development; and comparing the expression of thepolynucleotide to a normal standard wherein a higher than normalexpression indicates cancer or preneoplastic development in the tissueor organ in the suspect region.
 24. The method of claim 23, wherein thetissue or organ is selected from the group consisting of liver, breast,colon and kidney.
 25. The method of claim 24, wherein the tissue ororgan is liver.
 26. A method as claimed in claim 23 wherein theexpression of the polynucleotide of claim 1 is measured at the mRNAlevel.
 27. A method as claimed in claim 23 wherein the expression of thepolynucleotide of claim 1 is measured at the protein level.
 28. A methodfor identifying a human or non-human animal as a candidate for furtherscreening for cancer or preneoplastic development in a tissue or organ,the method comprising the steps of: determining the level of apolypeptide of claim 11 in a blood or blood-derived sample from theanimal; comparing the level to a normal range established by the sameanimal during a period that is tumor-free in the tissue or organ, or bya plurality of animals of the same species that are tumor-free in thetissue or organ; and identifying the animal as a candidate for furthercancer screening when the level exceeds the established normal range.29. The method of claim 28, wherein the tissue or organ is selected fromthe group consisting of liver, breast, colon and kidney.
 30. The methodof claim 29, wherein the tissue or organ is liver.
 31. A method foridentifying a human or non-human animal as a candidate for furtherscreening for cancer or preneoplastic development in a tissue or organ,the method comprising the steps of: determining the level of an antibodyto a polypeptide of claim 11 in a blood or blood-derived sample from theanimal; comparing the level to a normal range established by the sameanimal during a period that is tumor-free in the tissue or organ, or bya plurality of animals of the same species that are tumor-free in thetissue or organ; and identifying the animal as a candidate for furthercancer screening when the level exceeds the established normal range.32. The method of claim 31, wherein the tissue or organ is selected fromthe group consisting of liver, breast, colon and kidney.
 33. The methodof claim 32, wherein the tissue or organ is liver.
 34. A kit comprising:at least one of an antibody that specifically binds to a polypeptideconsisting of an amino acid sequence encoded by the first nucleotidesequence in claim 1, and a probe that hybridizes to the polynucleotidein claim 1; and at least one control sample component for which therelative or absolute amount of the polypeptide or polynucleotide isknown.
 35. A kit as claimed in claim 34 wherein the control samplecomponent is selected from the group consisting of liver cancer cells,preneoplastic liver cells, normal liver cells, breast cancer cells,normal breast cells, colon cancer cells, normal colon cells, kidneycancer cells, normal kidney cells, an extract of any of the foregoingcells, a blood sample from a human or non-human animal, and ablood-derived sample from a human or non-human animal.
 36. A kit asclaimed in claim 34, wherein the control sample component is an isolatedpolypeptide consisting of an amino acid sequence encoded by the firstnucleotide sequence in claim 1, or an isolated nucleic acid consistingof the polynucleotide in claim
 1. 37. A kit comprising: at least one ofan antibody that specifically binds to a polypeptide consisting of anamino acid sequence encoded by the first nucleotide sequence in claim 6,and a probe that hybridizes to the polynucleotide in claim 6; and atleast one control sample component for which the relative or absoluteamount of the polypeptide or polynucleotide is known.
 38. A kit asclaimed in claim 37, wherein the control sample component is an isolatedpolypeptide consisting of an amino acid sequence encoded by the firstnucleotide sequence in claim 6, or an isolated nucleic acid consistingof the polynucleotide in claim
 6. 39. A kit as claimed in claim 38,wherein the isolated polypeptide is selected from the group consistingof SEQ ID NO:2 and SEQ ID NO:4, and the isolated nucleic acid isselected from the group consisting of nucleotide 25 to nucleotide 1341of SEQ ID NO:1 and nucleotide 1 to nucleotide 1200 of SEQ ID NO:3.